Antibody-based therapies have gained importance in a variety of medical fields and have emerged as the most promising therapeutic approach in oncology. Antibodies against extracellular, cell surface associated, or secreted antigens associated with specific disease conditions are potentially of diagnostic, prognostic, and or therapeutic value. It has been shown that the therapeutic administration of monoclonal antibodies (mAb) directed against proteins associated with diseases is an effective therapy method of acute and chronic diseases such as cancers or rheumatoid arthritis. Examples for mAb targeted structures are the soluble protein tumor necrosis factor alpha (TNF-α) for rheumatoid arthritis, Crohn's disease and psoriasis (mAb preparation: Infliximab and Adalimumab), as well as the cell surface proteins CD20 for non-Hodgkin lymphoma (mAb preparation: e.g., Rituximab) and HER2/neu receptor (mAb preparation: Trastuzumab [Herceptin]) for breast cancer.
The development of the monoclonal antibody (mAb) technology represented a considerable achievement and resulted in numerous applications. However, in the field of immunotherapy, rodent mAbs have proved to be of limited use because of their strong immunogenicity in humans. Due to their low immunogenicity in patients, fully human mAbs are becoming increasingly important for the treatment of a growing number of diseases, including cancer, infectious disease, and immune disorders such as autoimmune diseases. The generation of monoclonal immunotherapeutically effective antibodies (using hybridoma or phage display techniques and subsequent chimerization and humanization, respectively), however, is time consuming and cost intensive which has prevented a broad clinical application so far.
Thus, there is a need for tools in the field of antibody-based immunotherapy which allow for the generation of fully human antibodies, preferably monoclonal antibodies, recognizing an antigen of interest in an easy, time and cost saving manner.
For full activation, B cells require two independent signals (FIG. 1). The first signal is antigen-specific and is mediated by the B cell receptor (BCR) recognizing its antigen. The BCR specifically binds the antigen and induces by receptor-clustering a signal-transduction cascade which leads to the transcriptional activation of genes associated with B cell activation. Upon BCR internalization, the antigen is processed and presented on MHC class II molecules. T cells which recognize the antigen in the context of the MHC class II molecule express CD40L on their surface and thus provide the second signal required for B cell activation, the stimulation of CD40 localized at the plasma membrane of B cells with its ligand CD40L. Activation of B cells results in the proliferation, differentiation, and antibody secretion.
The present invention provides tools for the isolation of antigen-specific B lymphocytes which is based on the antigen-specific expansion of a certain population of B lymphocytes. The present invention provides the possibility to imitate the two activation signals in vitro without the need for T cell co-stimulation. By transfection of a large number of B cells with the recombinant protein of the present invention and contacting the B cells with an antigen of interest, the B cell repertoire of a subject, for example, of a patient, can be screened for B cells having a defined antigen-specificity. The present invention allows for screening of a polyclonal B cell population and activation of monoclonal B cells and thus for the generation of antibodies, preferably human antibodies, which are specific for an antigen of interest.